Treatment of equine laminitis

ABSTRACT

Compositions and methods of the topical treatment of equine laminitis are disclosed. In particular, combinations of a fast acting nitric oxide (NO) donor, a sustained acting NO donor and an NSAID mixed in a lipid-based carrier are described. The application of such combinations to the affected areas, e.g., the hoofs and surrounding tissues, of an equine afflicted with laminitis provides relief from the debilitating effects of this painful, often life-threatening condition.

1. FIELD OF THE INVENTION

This invention relates to the treatment of equine laminitis, a verypainful condition affecting the laminae of an equine. Laminitisparticularly affects the area where the hoofs join the limbs, and canlead to foundering or lameness. Gone untreated, the condition can leadto the separation of the hoof from the bone and to a deterioration of anequine's overall health, including failure of internal organs followinginfection. The invention relates more particularly to a composition thatmay be topically applied to equine hoofs and surrounding tissue andwhich has an unusual curative effect on circulation throughout the area.A composition and method of alleviating the negative or adverse effectsof equine laminitis is, therefore, provided.

2. BACKGROUND OF THE INVENTION

Equine laminitis, which is sometimes referred to as foundering, is acommon disorder that has been recognized and described in even theearliest books of veterinary medicine. In an article by Green, M. E. etal., which appeared in Equine Medicine and Surgery, Fourth Edition, Vol.II, Colahan, P. T. et al. (Eds.), American Veterinary Publications, Inc.(1991), Chapter 12, pp. 1354-1358, laminitis is described as aninflammation of the pedal laminae that form the supportive bond betweenthe hoof and the third phalanx. Laminitis is further described as adisorder of the hoof whose cause is varied. It is a complex,multi-systemic disease affecting the digestive, cardiovascular, hemic,renal, endocrine, musculoskeletal, integumentary, and immune systems. Itis characterized by multi-systemic aberrations that ultimately result inreduced capillary perfusion, ischemia, and necrosis of the laminae.These results are accompanied by pain and loss of supportive function.Acute laminitis is described as comprising the events leading up to andthe onset of lameness. Acute laminitis can progress to the chronicstage. The chronic stage ensues after persistent lameness (greater than48 hours), or when the distal phalanx deviates detectably. Chroniclaminitis is a consequence of some degree of loss of integrity of thesupporting digital laminae. A photograph of a horse suffering from theeffects of laminitis of the forelimbs is shown on FIG. 1. Notice thatthe horse assumes a recumbent position in which most of the weight isplaced on the hind limbs and little weight bearing pressure is exertedon the forelimbs.

There are reports of evidence that equine laminitis is caused byingestion of too much grain; colic; retained placenta; exhaustion;ingestion of black walnut shavings; ingestion of too rich grass;excessive concussion, and/or excessive cold water. Despite the gatheringof voluminous information over a period exceeding 300 years, equinelaminitis still remains incompletely understood, however.

While it is possible that an equine that has been subjected to thiscondition can recover without intervention treatment, during the courseof the disease there is substantial pain, recumbency, hoof walldeformation and even sloughing of the hooves. Several symptoms aredisplayed, including: a bounding digital pulse, warm feet, an abnormalgait, a shifting of weight, or some combination of some or all of these.If the animal does not recover, after the onset of lameness, the laminaedeteriorate, the animal's feet are extremely painful, and the coffinbone becomes displaced. Destruction of the animal is then the onlyhumane course of action.

Because of the serious consequences of this condition, it has been thesubject of many and varied treatments over the years. In almost allcases, since the condition appears to be a function of a loss ofcirculation in the hoof area, the treatments have been directed toincreasing the blood supply to and circulation within the hoof andadjacent tissues.

There are several different treatments that are currently being used tocare for horses with laminitis. Several different vasodilators have beenused in the past to treat this disorder and some are still in use. Manyof them involve the use of nitroglycerin, which has been appliedtransdermally. Other methods use orally administered isoxsuprine, also aknown vasodilator. Current therapy also includes the use ofanticoagulants, such as heparin, aspirin and trental (pentoxifylline).All current therapies suffer from one or more drawbacks, includingdifficulty in the mode of administration of the active drug, lack ofeffectiveness, lack of compliance and lack of simplicity in the proposedtreatment regimen.

Katsuki, S. et al., in J. Cyclic Nucleotide Res. (1977) 3:23-35, pointout that the activity of a variety of smooth muscle relaxing agents,including sodium nitroprusside, nitroglycerin and sodium nitrite, may berelated to their ability to increase tissue levels of cyclic guanosinemonophosphate (cGMP) or to the formation of nitric oxide (NO). Othernitrogen-containing compounds may also function similarly, includinghydroxylamine or sodium azide. These substances may also possessvasodilatory properties. These and other nitrogen-containing compoundsmay then be referred to as nitric oxide "donors" or "precursors."

It has been shown that vascular endothelial cells synthesize nitricoxide from L-arginine (L-Arg) but not D-arginine. See, .Palmer R. M. J.et al., in Nature (1988) 333:664-666. This article also describes howthe release of NO from endothelial cells induced by bradykinin and thecalcium ionophore A23187 is reversibly enhanced by infusions of L-Arg orL-citrulline. The release of NO by certain cells can be protracted.Hence, L-Arg can be considered an NO donor or precursor, but because theconversion of L-Arg may not immediate and may occur over a protractedperiod of time, L-Arg can be thought of as a "slow acting" NO precursor.As used herein a "slow acting" NO precursor may provide for the releaseof NO in the tissues over the course of a few hours to several hoursafter initial exposure to the "slow acting" NO precursor. In contrast, a"fast acting" NO precursor, such as nitroglycerin or nitroprusside,generally provides almost instantaneous release of detectable levels ofNO in the plasma or tissue (e.g., within about a few minutes of exposureto the "fast acting" NO precursor and lasting for several minutes). Such"fast acting" agents may be quickly depleted, however. Thus, a fastacting NO donor may be used to provide a burst of NO, while a slowacting NO donor may be used to provide a more sustained, protractedlevel of NO release.

An abstract by Hinckley, K. A. et al., which appeared in J. Endocrinol.(1994) 143:P103, illustrates the complexity of the aetiology oflaminitis. These authors conclude that the causes of laminitis aremultifactorial. These authors speak only of the intravenousadministration of L-Arg.

In an editorial leader in the publication, Equine Vet. J. (1996)28(1):1-2, Elliott, J. discusses the merits of nitric oxide treatment oflaminitis. In this article, it is pointed out that NO was formerly knownas endothelium derived relaxing factor, a tribute to the fact that thisgas is produced continuously by the lining of blood vessels as a resultof the action of an enzyme present in the endothelial cells, endothelialnitric oxide synthase or eNOS. It is thought that eNOS is activated by arise in intracellular calcium concentration, catalyzing the conversionof L-Arg to NO and L-citrulline. The nitric oxide passes throughbiological membranes, binds to haem iron in the soluble enzyme guanylatecyclase (GC). The activity of GC is thus stimulated, cyclic guanosinemonophosphate concentrations increase and vascular smooth muscle tone isreduced. There is speculation in this article that disruption of theblood flow to the sensitive laminae of the equine foot, which occurs inlaminitis, might involve some disturbance in the L-Arg-NO pathway.

The subject of the editorial, an article by Hinckley et al. in the sameissue, Equine Vet. J. (1996) 28(1):17-28, describes a treatment thatuses intravenously administered L-Arg and topically appliednitroglycerin (glyceryl trinitrate or GTN). The L-Arg is administeredi.v. as a 10% aqueous solution, while the GTN is provided as a 2%ointment through a patch that is positioned, using adhesive tape, overdigital vessels. In particular, a laminitic pony, weighing 250 kg,received a total dose of 120 g of L-Arg or 0.48 g/kg bwt at a rate ofapproximately 40 mL/min (16 mg/kg bwt/min) for 30 min. The GTN patcheswere also applied once daily to three limbs only, 12 hours after theL-Arg infusion. Drops in heart rates were observed, includinghypotension and cardiac arrhythmia, with the i.v. infusion. Some poniesshowed signs of pain, sweating and shivering. The topical application ofGTN patches appeared to improve the lameness of some of the treatedponies. No improvement was observed in others. Thus, it is clear thatL-Arg, given intravenously, is not a particularly effective treatment.The results of the balance of this study are also mixed. There is nosuggestion in this article that L-Arg should be administered in a mannerother than intravenously or that any benefits may be obtained from afurther pursuit of a combination treatment.

Lecithin organogels are obtained by adding a minimal amount of water toa solution of lecithin in organic solvent. Typically, a minimum amountof water is added to lecithin dissolved in an organic solvent. See,Scartazzini, R. and Luisi, P. L., in J. Phys. Chem. (1988) 92:829-833.The results of an investigation on the transdermal transport ofscopolamine and broxaterol through human skin using a soybean lecithinorganogel has been described. Willimann, H. et al., in J. PharmaceuticalSci. (1992) 81(9):871-874. It was found that the rate of transdermaltransport of scopolamine was about one order of magnitude higher withthe organogel relative to an aqueous solution of the drug at the sameconcentration. However, the rate of transport of the drug was notdifferent with the organogel compared with a pre-gelation, microemulsionof the drug (lecithin, organic solvent and drug, but no added water).

In U.S. Pat. No. 4,882,164, granted to Ferro, A. and Steffen, H., anaqueous mixed micelle solution composed of cholanic acid salts andlipids is used for the solubilization of non-steroidal anti-inflammatorydrugs (NSAIDs). Cholanic acid salts include cholates, glycocholates andtaurocholates. Lipids include phosphatidylcholines, especially, naturaland synthetic lecithins. A formulation in accordance with the inventionis described, as follows: carprofen (50 mg), L-Arg (30 mg), anhydrousglycocholic acid (88.5 mg), 40% sodium hydroxide (19 microliters),lecithin for mixed micelles (169 mg), 2N HCl (to pH 6.0) and water forinjection (to make 100 mL final volume). The presence of the cholanicacid is essential to the composition described. Further, the presence ofan NO precursor or vasodilator is not disclosed, taught, or suggested.

NSAIDs are compounds that are structurally different from steroids andwhich display an anti-inflammatory activity. NSAIDs usually contain acarboxylic acid group and are derived from acetic acid, propionic acid,butyric acid, salicylic acid and the like. Subcategories of NSAIDs,include aminoarylcarboxylic acids (e.g., enfenamine, flufenaminic acid,isonixin, niflumic acid and the like), arylacetic acids (e.g.,acemetacin, alcoflenac, amfenac, clopirac, felbinac, fenclofenac,ibufenac, indomethacin, isoxepac, sulindac, zomepirac and the like),arylbutyric acids (e.g., bumadizon, fenbufen, xenbucin and the like),arylcarboxylic acids (e.g., clinadac, ketorolac and the like),arylpropionic acids (e.g., benoxaprofen, carprofen, ibuprofen,ibuproxam, indoprofen, ketoprofen, naproxen, oxaprozin, protizinic acid,suprofen and the like), pyrazoles, pyrazolones and thiazinecarboxamides.Specific examples of NSAIDs include, but are not limited to, flunixin,epirizole, apazone, feprazone, ramifenazone, droxicam, aspirin,phenylbutazone, piroxicam, bendazac, ditazol, guaiazulene, oxaceprol,proxazole, tenidap and the like, or the salts thereof.

Hence, a strong need remains for an effective treatment of equinelaminitis.

3. SUMMARY OF THE INVENTION

In accordance with the invention, a topical composition for thetreatment of equine laminitis is described comprising a fast actingnitric oxide (NO) donor, preferably nitroglycerin, and a sustainedacting NO donor, preferably L-arginine (L-Arg), dispersed in alipid-based carrier. In a preferred embodiment of the invention, thetopical composition further comprises a non-steroidal anti-inflammatorydrug (NSAID). The lipid-based carrier comprises preferably a membraneforming lipid, such as a phosphatidylcholine (e.g., a naturallyoccurring, naturally derived, synthetic, or semi-synthetic lecithin).Preferably, the topical combination is in the form of an ointment,emulsion, creme, gel, or foam that permits the delivery of the activeingredients of the fast acting nitric oxide donor and L-Arg through thehoof, skin, or both of an affected equine.

The invention also relates to a method of ameliorating the adverseeffects of equine laminitis. The method, broadly contemplated, comprisestopically administering to the affected areas of an equine an effectiveamount of a fast acting nitric oxide (NO) donor dispersed in alipid-based carrier. In a preferred embodiment, the method furthercomprises topically administering to the affected areas an effectiveamount of a sustained acting NO donor. Most preferably, the method evenfurther comprises topically administering to the affected areas aneffective amount of a non-steroidal anti-inflammatory drug (NSAID). Theforegoing active ingredients can be administered in any sequence (i.e.,sequentially) or substantially at the same time period (i.e.,contemporaneously).

By utilizing the compositions and methods of the present invention, ithas surprisingly been discovered that topical application directly tothe affected areas of an equine afflicted with the adverse effects ofequine laminitis, usually suffering from extreme pain, lameness,foundering, even partial separation of the hoof from the limb, reducedor failing organ function, poor appetite and near death, providesastounding results. In particular, treated horse or ponies exhibitremarkable signs of recovery, including regaining the ability to stand,improved posture, normal gait, improved appetite, or improved and/orstabilized organ function and generally a return to normal activities.

The present invention also contemplates a process for the preparation ofa combination, which can be applied topically to alleviate the adverseeffects of equine laminitis, comprising: (a) combining a first mixturecomprising at least one alcoholic solvent, at least one fast actingnitric oxide (NO) donor, at least one sustained acting NO donor and atleast one NSAID with a second mixture comprising at least one membraneforming lipid and at least one biocompatible organic solvent; (b)admixing to the resulting mixture of step (a) an amount of a thirdmixture comprising water and a surfactant effective to provide acombination having a creamy texture.

It is therefore a primary object of this invention to provide a noveltreatment for equine laminitis.

It is another object of this invention to provide a novel compositionthat is suitable for topical application as a treatment for equinelaminitis.

It is a further object of this invention to provide a method of treatinglaminitis comprising topically applying an effective amount of acombination or composition that is effective for the treatment of equinelaminitis.

Other and additional objects will become apparent to those of ordinaryskill from a consideration of general and specific descriptions providedin this specification.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a pony suffering from the adverse effects of equinelaminitis (recumbent).

FIG. 2 illustrates the pony of FIG. 1 after responding to treatmentusing the composition and method of the present invention (note straightfront legs).

FIG. 3 illustrates the recovered pony of FIG. 2 engaged in normalactivities, such as eating grass, while placing weight normally on theforelimbs and hind limbs (12 weeks from FIG. 1).

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

5.1. Glossary

Nitric Oxide (NO) Donor--Any substance that is converted into, degradedor metabolized into, or provides a source of in vivo nitric oxide or NO.A "fast acting" NO donor is a nitric oxide donor that gives rise to thein vivo production of NO in or around the affected areas, tissues,hoofs, limbs, etc. of the afflicted equine within about one minute towithin about ten minutes of topical application of the fast acting NOdonor on the affected areas, tissues, hoofs, limbs, etc. of theafflicted equine. A "sustained acting" NO donor is a nitric oxide donorthat gives rise to the in vivo production of NO in or around theaffected areas, tissues, hoofs, limbs, etc. of the afflicted equinewithin about thirty minutes to within about a few to several hours(e.g., two, four, six, or eight hours) of topical application of thesustained acting NO donor on the affected areas, tissues, hoofs, limbs,etc. of the afflicted equine.

Lipid-Based Carrier--Any carrier, preferably a pharmacologicallyacceptable carrier, which is comprised of lipid or fatty components,especially those having a hydrophobic moiety and a hydrophilic moiety,and which enables the transdermal transport of an active ingredient fromthe surface of the skin to the regions of the body below the skin. Oncebelow the skin, the active ingredient(s) may remain localized in oraround the region(s) to which the combination or composition ofinterest, which is partially comprised of the lipid-based carrier, hasbeen applied. Alternatively, the active ingredient(s) may becomeavailable systemically. A preferred class of lipids, include those thatform membranes, bilayers, vesicles, liposomes and the like, particularlybiological membranes. Examples of such membrane-forming lipids includebut are not limited to phospholipids, glycolipids and cholesterol-typelipids. Preferred lipids include phosphatidyl choline, phosphatidylserine, phosphatidyl inositol, phosphatidyl inositol and the like.

Biocompatible Organic Solvent--An organic solvent, typically comprisedof esters of fatty acids. The fatty acids contain long chain saturatedor unsaturated aliphatic groups having about 8-50 carbon atoms,preferably about 12-30, more preferably about 14-24 carbon atoms, mostpreferably about 16 to about 18 carbon atoms. Examples of fatty acidsinclude lauric, myristic, palmitic, stearic, arachidic, behenic,lignoceric, palmitoleic, oleic, linoleic, linolenic, arachidonic acidand the like. The alcohol portion of the ester group is generally alinear or branched lower alkyl group comprising about 1-8 carbon atoms.Examples of the alcohol portion include methanol, ethanol, propanol,isopropanol, butanol, isobutanol, sec-butanol, tert-butanol, pentanol,isopentanol, hexanol, heptanol, octanol, isooctanol, cyclooctanol andthe like. Accordingly, preferred esters include but are not limited toethyl palmitate, ethyl myristate, ethyl oleate, isopropyl palmitate,isopropyl myristate, isopropyl oleate, and the like. Other biocompatibleorganic solvents may include isooctane and cyclooctane.

Second Hydroxyl Group Containing Aliphatic Organic Solvent--An organicsolvent containing a hydroxyl group, including but not limited to thosedescribed in the preceding paragraph relating to the "alcohol portion ofthe ester group."

Surfactant--Any compound or surface active agent that reduces surfacetension when dissolved in water or water solutions, or which reducesinterfacial tension between two liquids, or between a liquid and asolid. Surfactants suitable for use in the present invention includeionic and non-ionic detergents, dispersing agents, wetting agents,emulsifiers and the like. Examples of surfactants include but are notlimited to the sodium salt of an N-(alkylsulfonyl)glycine (EMULSIFIERSTH), the salts of linear alkyl sulfonates (LAS), the salts of alkylbenzene sulfonates (ABS), the sodium salt of dodecylsulfate (SDS), anonionic series of 28 related difunctional block-polymers terminating inprimary hydroxyl groups with molecular weights ranging from about 1,000to over about 15,000, polyoxyalkylene derivatives of propylene glycol(PLURONIC), and the like. The surfactant used in the present inventionis preferably pharmaceutically acceptable and biodegradable.

5.2. The Topical Composition

As described above, the present invention is directed to a topicalcomposition for the treatment of equine laminitis. In a particularembodiment of the invention, the topical composition comprises a fastacting nitric oxide (NO) donor and a sustained acting NO donor. In apreferred embodiment, the topical composition further comprises anon-steroidal anti-inflammatory drug (NSAID). For maximizing theeffectiveness of the topical composition of the present invention, theactive ingredients, such as nitroglycerin, L-Arg and ketoprofen (a fastacting NO donor, a sustained acting NO donor and an NSAID, respectively)are dispersed, optionally with other active or non-active ingredients,thickeners, emollients, fillers, etc., are dispersed in a lipid-basedcarrier.

The lipid-based carrier is preferably a membrane forming lipid, such asphosphatidyl choline, phosphatidyl serine, phosphatidyl inositol,phosphatidyl ethanolamine, or the like. The preferred membrane lipidcomprises lecithin, as already described above. Hence, in a particularembodiment of the invention, the lipid-based carrier comprises at leastone membrane forming lipid, at least one first biocompatible aliphaticorganic solvent comprising 8-50 carbon atoms, at least one secondhydroxyl group containing aliphatic organic solvent comprising 1-8carbon atoms and at least one surfactant.

Typically, phospholipids suitable for the present invention are derivedfrom glycerol (e.g., a phosphoglyceride), sphingosine, or other simpleor complex alcohols, including but not limited to serine, glucose,galactose, ethanolamine, choline, inositol, mannitol and the like. Thefatty acid portion of the lipid may comprise typically of about 14 toabout 24 carbon atoms, more typically about 16 to about 18 carbon atoms,in particular, palmitate, oleate, or myristate. Preferred lipids includesphingomyelin, cerebroside, cholesterol, cholic acid, or phosphatidylcholine, phosphatidyl ethanolamine and the like. More preferably, thelipid-based carrier is comprised of lecithin, ether naturally occurring,naturally derived, synthetic, or semi-synthetic. A pure lecithin iscomprised of a phosphatidyl choline. That is, lecithins are mixtures ofdiglycerides of fatty acids linked to the choline ester of phosphoricacid. Lecithins may also be classified as phosphatides, as well asphosphoglycerides.

A fast acting NO donor according to the invention may be selected fromnitroglycerin, hydroxylamine, a nitrite, a nitroprusside, an azide, or asalt thereof. The sustained acting NO donor according to the inventionmay in turn be selected from L-Arg, derivatives thereof, analogs thereofand the like.

Particular NSAIDs suitable for use in the present invention includethose derived from acetic acid, propionic acid, butyric acid, salicylicacid and the like. Also, suitable are those NSAIDs derived from anaminoarylcarboxylic acid, arylcarboxylic acid, arylacetic acid,arylpropionic acid, arylbutyric acid, pyrazoles, pyrazolones,thiazinecarboxamides and the like. Most preferably, the NSAID selectedis ketoprofen, ibuprofen, or naproxen. Additional NSAIDs are describedin the Background Section, above.

In a preferred embodiment of the invention, the topical compositioncomprises about 0.5 to 3% by weight nitroglycerin and about 6 to 20% byweight of L-Arg, based on the weight of the total composition. Morepreferably, the topical composition comprises about 1.0 to 2.4% byweight nitroglycerin and about 10 to 15% by weight of L-Arg, based onthe weight of the total composition. In yet another embodiment, thetopical composition comprises about 5 to 16% by weight nitroglycerin,based on the weight of L-Arg present in the total composition, morepreferably about 7 to 8% by weight nitroglycerin, based on the weight ofL-Arg present in the total composition. Where the topical combinationincludes an NSAID, the NSAID preferably is present in an amount that isup to about 10% by weight, based on the weight of the total combination.Other active ingredients, which may also be present in the topicalcombination, include analgesics, topical anesthetics and the like.

As described above, the topical composition of the invention may come inany form, but preferably as an ointment, emulsion, creme, gel, or foam.What is important, however, is that the lipid-based carrier permits thedelivery of the fast acting nitric oxide donor, L-Arg and otherprincipal active ingredients through the hoof, skin, or both of anaffected equine. In a specific embodiment of the invention, thelipid-based carrier further comprises at least one pharmaceuticallyacceptable alcohol.

Hence in a particular embodiment a topical composition is provided forthe amelioration of the negative effects of equine laminitis comprisingat least one fast acting nitric oxide (NO) donor, L-Arg and at least oneNSAID dispersed in a lipid-based carrier comprising at least onemembrane forming lipid, at least one first biocompatible aliphaticorganic solvent comprising 8-50 carbon atoms, at least one secondhydroxyl group containing aliphatic organic solvent comprising 1-8carbon atoms and at least one surfactant.

5.3. The Topical Administration

The present invention is directed to a method of alleviating orameliorating the adverse or negative effects of equine laminitis.Generally, the desired method comprises topically administering to theaffected areas of an equine an effective amount of a fast acting nitricoxide (NO) donor dispersed in a lipid-based carrier. Other methods ofincreasing desirability include the topical administration to theaffected areas an effective amount of a sustained acting NO donor and,also, the topical administration to the affected areas an effectiveamount of a non-steroidal anti-inflammatory drug (NSAID). Preferably, atopical composition containing a fast acting NO donor, a sustainedacting NO donor and an NSAID is applied daily for a period of at leastabout a few weeks (e.g., for at least about one week, two weeks, threeweeks, or four weeks) to a few months (e.g., for at least about onemonth, two months, or three months) to the affected areas of the equine.It is important to point out, however, that each of the principal activeingredients can be topically administered sequentially or more or lesscontemporaneously. Also, the topical composition can be applied oncedaily or more than once, for example, twice daily or as needed.

The daily dosage of the fast acting NO donor ranges from about 0.165 toabout 0.7 mg/kg of the equine, preferably from about 0.2 to about 0.5mg/kg of the equine, most preferably from about 0.3 to about 0.4 mg/kgof the equine. In a specific embodiment of the invention, the dose offast acting NO donor is about 0.33 mg/kg of the equine. The daily dosageof the sustained acting NO donor ranges from about 4 to about 9 mg/kg ofthe equine, preferably from about 5 to about 8 mg/kg of the equine, mostpreferably about 6 to about 7 mg/kg of the equine. The daily dosage ofthe NSAID ranges from about 1.4 to about 3.2 mg/kg of the equine,preferably from about 1.7 to about 3.0 mg/kg of the equine, mostpreferably from about 2.0 to about 2.8 mg/kg of the equine.

When the fast acting NO donor comprises nitroglycerin and the sustainedacting NO donor comprises L-Arg, the preferred dosages range from about0.3 to about 0.4 mg/kg of the equine and from about 6.5 to about 7 mg/kgof the equine, respectively.

In a specific embodiment of the invention a method is provided ofameliorating the adverse affects of equine laminitis which comprisestopically administering at least once daily to the affected areas of anequine an effective amount of a combination comprising at least one fastacting NO donor, L-Arg and at least one NSAID in a lipid-based carrier;and continuing to administer said combination until the equine exhibitssigns of recovery. Such signs of recovery include but are not limited toregaining the ability to stand, improved posture, normal gait, improvedappetite, or improved or stabilized organ function. Still other signsmay be obtained from performing blood tests on the affected equine,including a horse, pony, donkey, ass, zebra and the like. For examplethe blood test could show normal creatinine or blood urea nitrogenlevels, where these levels would be elevated in ill equines.

5.4. The Process of Preparing the Topical Composition

The present invention is also directed to a process for the preparationof a combination, which can be applied topically to alleviate theadverse effects of equine laminitis. In particular, the topicalcombination or composition is obtained by first preparing a firstmixture comprising at least one alcoholic solvent, at least one fastacting nitric oxide (NO) donor, at least one sustained acting NO donorand at least one NSAID. A second mixture is then prepared, whichcomprises at least one membrane forming lipid and at least onebiocompatible organic solvent. The first and second mixtures are thencombined with mixing, preferably at or slightly below room temperature.

To the resulting mixture, which contains the principal activeingredients (e.g., fast acting NO donor, sustained acting NO donor,optionally with an NSAID), an amount of a third mixture comprising waterand a surfactant is then added, which amount is effective to provide acombination having a creamy texture. The preferred surfactant isPLURONIC.

5.5. More General Aspects of the Preferred Embodiments

As stated above, in general, the contemplated composition of thisinvention comprises a combination of L-Arg and nitroglycerin suitablyput up in the form of a creme or ointment. It may also be used in theform of a foam. Suitable foams, ointments and cremes of topicallyapplied therapeutic compositions are available in the art. Specifically,the topically applied compositions of this invention comprise the activeingredients as aforesaid and in addition comprise non-active components,such as a suitable carrier, which may be water-based or organic-based ora combination of both.

An ingredient in compositions, which are intended for topicalapplication, may be a material that assists in causing the activeingredients to pass through the dermal layer to permit the subcutaneousattack of the condition sought to be ameliorated. Such dermal layerpenetration assistants, which have been found to be useful in thisinvention, include membrane forming lipids and alcohols, particularlywater soluble alcohols, and other mutual solvents that cause thenormally water insoluble active ingredients to have increased watersolubility and therefore increased dermal penetrability. Ethanol is onesuch suitable material, and is the dermal penetration assistant materialof choice because it is readily available, is inexpensive and it ispharmaceutically acceptable. Ethanol is also the preferred solvent fordissolving the principal active ingredients of the present topicalcompositions for incorporation into the lipid-based carrier.

Another common constituent of topically applicable compositions is anemollient. Isopropyl palmitate is one example of such emollientmaterials. Other such materials are well known in this art and will beapparent to those of ordinary skill. Surfactants are common ingredientsin cremes and ointments for transdermal delivery of medicaments.Non-ionic, cationic and anionic surfactants are all considered to besuitable for use in the preparation of the topically applied compositionof this invention. One suitable surfactant is a commercially availablematerial sold under the name PLURONIC. Others will be known to theperson of ordinary skill in this art.

It is considered to be within the scope of this invention to provideadditional therapeutic agents in the topically applied compositionhereof in order to treat specific conditions that often accompany equinelaminitis. This condition is painful. It is therefore appropriate toinclude topical anesthetics in the inventive compositions. Topicalanalgesics are suitable constituents as well. NSAIDs, such as ketoprofenand naproxen, are useful additions to the creme and/or ointmentcompositions of this invention. Further, thickening agents are a usefulpart of the compositions of this invention. Hydroxymethyl orhydroxyethyl cellulose and the like can be used as thickening agents.

Cremes and ointments are known to require a combination of ingredientsto balance the physical properties and to act as an effective carrierand delivery system for the medicaments that are incorporated therein.Where the topically applied composition of this invention is put up inthe form of a foam, it is suitable to include conventionalpharmaceutically acceptable blowing agents in the product. Suitablybutane, propane, pentane and mixtures thereof have been found to beuseful. These gases are relatively inert materials that have been usedto make foams in the past, and they will be useful in the invention.Suitably, the volatile compound, such as a light hydrocarbon or carbondioxide, is incorporated into the composition of this invention in apressurized contained, such as a conventional aerosol can. When thecomposition is released, the light hydrocarbon blowing agent expands andconverts the composition of this invention into a foam for topicalapplication.

The active ingredients of the topically applicable compositions of thisinvention, illustrated by nitroglycerin and L-Arg, are suitably used inamounts of about 0.5 to about 3% by weight of nitroglycerin and about 5to about 25% by weight of L-Arg, based on the weight of the entirecomposition. It is preferred to use about 1 to about 2.5% by weight ofthe nitroglycerin and about 15 to about 20% by weight of the L-Arg. Theweight ratio of fast acting NO donor to sustained acting donor rangesfrom about 1:5 to about 1:50 for the total composition. There ispreferably used about 2 to about 10% by weight of NSAID, preferablyabout 4 to about 8%, based on the weight of the total composition.Hence, the weight ratio of NSAID to sustained acting NO donor rangesfrom about 1:1.5 to about 1:5. The total amount of principal activeingredients can range from about 7.5 to about 45% by weight, preferablyabout 15 to about 35%, most preferably about 20 to about 30%, based onthe total composition.

The ointment or salve or lotion or foam, which is to be topicallyapplied, suitably contains up to about 23% by weight of the specificcombination of nitroglycerin and L-Arg active ingredients, based on thetotal weight of the fully formulated topical composition, preferably upto about 16% by weight, most preferably up to about 14% by weight.

As indicated above, the effective topically applicable composition ofthis invention comprises a combination of a fast acting NO donor, suchas nitroglycerin, azide, hydroxylamine and the like, and a moresustained, slower acting NO donor, such as L-Arg, in the proportions setforth above. When this active composition is in the form of a creme orointment, the other conventional components of the cream or ointmentinclude a carrier, such as water or mineral spirits, which will be themain or principal ingredient of the composition when considered on aweight or volume basis. This carrier or solvent will be present in thecomposition in amounts of about 40 to 90 weight percent of the wholecomposition, preferably about 60 to about 85 percent, more preferablyabout 70 to about 80 weight percent. Emollients, such as lecithin, orthe like, are used in a proportion of up to about 10 weight percentbased on the weight of the entire composition.

Conventional odorants, such as perfumes, and colorants, such as fooddyes, can be used in their conventional proportions for theirconventional purposes. Where needed, deodorants can also be used.

The proportion of the carrier materials, such as water, or a mixture ofwater and an organic solvent, such as an alcohol, will be theconventional amount that will make a smoothly applicable creme orointment that remains on the dermal surface for a time sufficient toenable substantially all of the active ingredients to pass out of thetopical application composition and pass into active relationship withthe blood vessels of the equine hoofs.

Nitroglycerin and L-Arg have been specified as the active ingredients ofthe compositions of this invention. It will be understood thatnitroglycerin is a fast acting NO donor, an immediate vasodilator, whileL-Arg must first be converted to NO before it becomes effective.Therefore, the L-Arg has a more sustained release. Other immediate andsustained release sources of NO are believed effective in combatingequine laminitis by means of a topically applied composition comprisingcombinations described herein. The compounds that appear to becomparable to nitroglycerin and L-Arg, in that they have known tendencyto release NO either rapidly/immediately, or slowly/sustained,respectively, are described herein. Others may be apparent to those ofordinary skill in the art given the descriptions provided herein. Thus,other vasodilators, such as isoxsuprine or nitroprusside may besubstituted for nitroglycerin. Other NSAIDs may be substituted for theketoprofen, such as phenylbutazone, aspirin, flurixin, ibuprofen andnaproxen. Slow/sustained acting NO releasing materials, which havesimilar activity to L-Arg, may also be apparent to those of ordinaryskill in the art considering the description presented herein.

It has been found, and this is another aspect of this invention, thatthe transdermal transport of pharmaceutically active components, such asthe NO donors of this invention, can be substantially improved. Theactive ingredients, that is the nitroglycerin, the L-Arg, or either ofthem, plus NSAID, are first dissolved in ethanol or some otherpharmaceutically acceptable solvent. Preferably this solvent is alsocompatible with water. This active ingredient containing solution isthen converted into the topical creme or ointment by being admixed orcombined with a lipid, surfactant, biocompatible organic solvent, water,thickeners, emollients, surfactants, and/or other conventionalingredients of a creme, ointment or foam. The resulting topicalcomposition is then applied to an affected portion of the dermal layer,such as a horse's hoofs and surrounding tissue. The ameliorating affectof the creme of the invention, so prepared, is quite dramatic and isillustrated further by the Examples provided, below.

6. EXAMPLES

The following are illustrative of the compositions and methods that arecontemplated by the present invention.

6.1. Preparation of PLURONIC Stock Solution

Potassium sorbate (NF, 0.3 g) and PLURONIC F127 (NF, 20 g and availablefrom PCCA, Houston, Tex.) are combined together with purified water(q.s., to a volume of 100 mL). The resulting 20% pluronic stock solutioncan be kept refrigerated. Thirty percent (30%) and forty percent (40%)stock solutions can also be prepared by using 30 g and 40 g of PLURONICF127, respectively.

6.2. Preparation of Lipid/Biocompatible Solvent Mixtures

Soya Lecithin (granular, 100 g) and sorbic acid (NF-FCC powder, 0.66 g)are dispersed in isopropyl palmitate (NF, 100 g or ca. 117 mL). Themixture is allowed to stand overnight to provide a syrupy liquid. Otherbiocompatible organic solvents can be used in place of isopropylpalmitate, so long as it dissolves the lecithin (e.g., commercial gradesoy or egg lecithin). Examples of other suitable organic solventsinclude, but are not limited to, isopropyl myristate (preferably,cosmetic grade) or cyclooctane.

6.3. Preparation of Lecithin PLURONIC Gel

A lecithin pluronic gel can be prepared by combining soy lecithin(granular, 10 g) with sorbic acid (NF-FCC powder, 0.3 g) in isopropylpalmitate (NF, 10 g or 11.7 mL). The mixture is allowed to standovernight to provide a syrupy liquid. Enough 20% pluronic stock solutionis then added to the syrup to brng the final to 100 mL. To the resultinglecithin pluronic gel is added L-Arg (25 g), nitroglycerin (0.8 g) andindomethacin (10 g). The resulting mixture is mixed thoroughly.

Alternatively, 52 g of soya lecithin/isopropyl palmitate solution ismixed with 19 g of 20% pluronic stock solution. To this mixture is addedethoxy diglycol (2 g), purified water (27 g), L-Arg (15 g), ketoprofen(6 g) and nitroglycerin (1.2 g). The resulting mixture is mixedvigorously until a creamy consistency is obtained.

6.4. Preparation of Transdermal Creme

Lecithin (10 g) dispersed in isopropyl myristate (10 g) is combined witha solution comprising L-Arg (15 g), ketoprofen (6 g) and nitroglycerin(1.2 g) dissolved in ethanol (10 mL). To this mixture is added anaqueous mixture of pluronic (20 g) in water (q.s., 120 g). The combinedingredients are mixed until a creamy consistency is obtained. Theresulting creme can be applied topically directly to the hoof andsurrounding tissue of an equine, such as a horse, to provide a treatmentfor alleviating the negative effects of equine laminitis:

Other suitable formulations are provided below:

    ______________________________________    Ingredients Amounts in Grams    Example No. 1      2       3    4     5    6    ______________________________________    L-Arg       25     25      15   25    25   15    ketoprofen  10     5       6    5     10   6    ethanol (mL)                20     20      10   20    10   10    lecithin    10     10      10   10    10   10    pluronic    20     20      20   20    20   20    nitroglycerin                0.6    1.2     1.2  1.0   1.2  2.4    isopropyl palmitate                10     10      10   10    10   10    water, q.s. (total g)                120    120     120  120   120  120    ______________________________________

Other NSAIDs, such as naproxen, ibuprofen and the like, can be used inplace of ketoprofen. Other fast acting NO precursors, such asnitroprusside, hydroxylamine and the like, can be used in place ofnitroglycerin. Other biocompatible organic solvents, such as isopropylmyristate, cyclooctane, isooctane and the like, can be used in place ofisopropyl palmitate.

6.5. Use of the Transdermal Creme on a Laminitic Pony

A foundered pony (laying down, unable to stand) is treated with about 5g of the transdermal creme of Example 3 from Section 6.4, above, threetimes daily. The third phalange or P-3 is observed through the sole onboth feet of the pony. The creme is applied to the coronary band and tothe area at the tip of the P-3 daily for a period of three to fourweeks. By about week 3, the pony is able to accept nailed-in shoes andstand straight up. This improvement is remarkable given the pony'sinitial breached position. By the end of the fourth week, the pony iswalking freely and eating normally.

A horse, shown in FIG. 1, is exhibiting a recumbent position because ofthe pain associated with laminitis of the forelimbs. This horse istested for liver and kidney function (e.g., creatinine,blood-urea-nitrogen (BUN) levels). The blood tests show greatlydiminished organ function (i.e., high levels of creatinine and BUN).After one month of treatment using the transdermal creme of Example 3from Section 6.4, the horse is able to stand upright (See, FIG. 2) andmove normally (See, FIG. 3). Organ function tests that are conductedafter the treatment period confirms that the horse's organs havereturned to normal or near normal function.

It should be apparent to one of ordinary skill in the art that otherembodiments can be readily contemplated in view of the teachings of thepresent specification. Such other embodiments, while not specificallydisclosed nonetheless fall within the scope and spirit of the presentinvention. Thus, the present invention should not be construed as beinglimited to the specific embodiments described above, which invention islimited solely by the claims to follow.

What is claimed is:
 1. A method of ameliorating the adverse effects ofequine laminitis comprising topically administering to the affectedareas of an equine an effective amount of a fast acting nitric oxide(NO) donor and a slow acting NO donor dispersed in a lipid-based carrierand continuing to administer said combination until the equine exhibitssigns of recovery, said recovery comprises regaining the ability tostand, improved posture, normal gait, improved appetite, or improved orstabilized organ function.
 2. The method of claim 1 which furthercomprises topically administering to the affected areas an effectiveamount of a non-steroidal anti-inflammatory drug (NSAID).
 3. The methodof claim 1 in which the fast acting NO donor and the slow acting NOdonor are topically administered sequentially.
 4. The method of claim 1in which the fast acting NO donor and the slow acting NO donor aretopically administered contemporaneously.
 5. The method as claimed inclaim 1 in which said fast acting NO donor comprises nitroglycerin andsaid slow acting NO donor comprises L-Arg.
 6. The method of claim 2 inwhich said NSAID is derived from an aminoarylcarboxylic acid,arylcarboxylic acid, arylacetic acid, arylpropionic acid, arylbutyricacid, pyrazoles, pyrazolones, or thiazinecarboxamides.
 7. The method ofclaim 1 which is applied daily for at least about one week, two weeks,three weeks, or four weeks.
 8. The method of claim 1 which is applieddaily for at least about a month.
 9. The method of claim 2 which isapplied daily for at least about a month.
 10. The method of claim 1 inwhich said lipid-based carrier comprises at least one membrane forminglipid, at least one thickening agent, at least one emollient and atleast one surfactant.
 11. The method of claim 1 in which saidlipid-based carrier further comprises at least one pharmaceuticallyacceptable alcohol.
 12. A method of ameliorating the adverse effects ofequine laminitis which comprises topically administering at least oncedaily to the affected areas of an equine a combination comprising atleast one fast acting NO donor at least one slow acting donor, L-Arg,and at least one NSAID in a lipid-based carrier; in an amount sufficientto promote signs of recovery, said recovery comprises regaining theability to stand, improved posture, normal gait, improved appetite, orimproved or stabilized organ function.
 13. The method of claim 12 inwhich a blood test shows normal creatinine or blood urea nitrogenlevels.